Abstract

Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER. (Cancer Res 2006; 66(17): 8590-7)

Introduction

DNA repair is the critical cellular process for preventing cell killing, mutations, replication errors, persistent DNA damage, and genomic instability. Abnormalities of DNA repair have been implicated in cancer and aging. Nucleotide excision repair (NER) is the most important DNA repair pathway that fixes the majority of bulky lesions in DNA. These lesions include UV-induced photoproducts and bulky adducts e.g., those arising from exposure to carcinogenic chemicals. NER includes two distinct subpathways, global genomic repair, which removes the lesions in the whole genome, and transcription-coupled repair, which eliminates the lesions located within the transcribed strands of transcriptionally active genes (
1). Basic NER contains three distinguishable segments—recognition of damage within the compact chromatin, incision, and excision of damage containing oligonucleotide and finally the gap-filling DNA synthesis and ligation (
2,
3). The biological consequences of NER defects are apparent from the inherited multisystem disorders, e.g., xeroderma pigmentosum (XP), which is characterized by hypersensitivity to sun light and extremely high incidence of cancer. XP-E is one of these XP forms and the cells from XP-E patients show the mildest deficiency in NER among all the XP patients.

XP-E cells lack UV-DDB activity associated with a damaged DNA-binding (DDB) protein complex (
4), which comprises two subunits, DDB1 (p127) and DDB2 (p48; refs.
5–
8). Despite the known ability of DDB protein to bind UV-irradiated DNA, the specific role of DDB in NER has only recently begun to take a clearer shape. The role of DDB2 subunit in NER has been extensively studied and the activity-altering sequence changes have been identified in the DDB2 gene in eight human XP-E cases thus far confirmed. On the other hand, no sequence changes have been found in DDB1 gene (
9–
11). Growing body of evidence has confirmed that DDB2 is undeniably involved in global genomic repair. For example, several studies showed that the cells from some XP-E patients have a partial deficiency in NER (
9,
12–
14). Microinjection of the purified DDB complex into XP-E cells reversed the NER defect (
11,
15,
16). Some rodent cell lines, such as Chinese hamster V79 cells, rarely express endogenous rodent DDB2, and show very slow removal of cyclobutane pyrimidine dimer (CPD) from the genome. This repair defect can be restored either by reactivating DDB2 gene expression, or by ectopic expression of human DDB2 in the cells (
9,
12,
13). NER can be reconstituted with purified components and damaged DNA in the absence of DDB. Adding DDB to the purified NER system, using a substrate with a single CPD lesion, has been shown to result in no stimulation (
17), to a few fold stimulation (
18), and even a 17-fold stimulation (
14). Therefore, at present, DDB is believed to be relevant only to the NER within the chromatin context and it could be having a minor accessory role in NER in vitro when chromatin-free DNA is the substrate (
19–
21).

DDB1 was first isolated, together with DDB2, as a subunit of a heterodimeric DDB protein complex that shows a high affinity to UV-damaged DNA (for a review, see ref.
22). DDB1 is a relatively abundant cellular protein and is present in excess over that of its critical partner, DDB2, in unirradiated HeLa cells (
5,
23). DDB1 is vital for normal cell function and evolutionarily conserved in mammals, worms, insects, and plants. To date, no deleterious naturally occurring mutations have been found in mammalian DDB1. More recently, DDB1 and DDB2 were found to form a complex with cullin 4A (Cul4A), which serves as an E3 ubiquitin ligase (
24–
26). Cul4A is a member of the cullin family of proteins. It has been proposed that Cul4A proteins function as E3 ligases, which are involved in selecting specific targets for ubiquitylation. However, unlike other ligases, Cul4A cannot bind to substrates directly, but requires a specificity factor to recruit target substrates to Cul4A. DDB1 is found to associate with Cul4A and considered to serve the function of such substrate-recruiting factor for Cul4A-Roc1 ligases (
24,
26–
28). In combination with Cul4A, DDB1 has been proposed to either directly dock a substrate to the E3 machinery or indirectly recruit a substrate through an additional adaptor(s) (
26). COP9 signalosome has also been found in complex with DDB1-Cul4A-Roc1, regulating the ubiquitin ligase activities of this complex (
25). DDB1 protein has lately attracted considerable interest because of its multiple functions in cells. However, the action of DDB1 in the context of DNA repair has received very little attention although DDB is now clearly implicated in NER and the role of its DDB2 component is reasonably well established. We hypothesized that DDB1 can contribute to NER through bridging Cul4A ligase and DDB2. To address this question, we used DDB1 small interfering RNA (siRNA) to abrogate the expression of DDB1 in the mammalian cells and investigated the effect of DDB1 deficiency on the removal of UV-induced photolesions as well as the recruitment of DDB2 and Cul4A to such damage sites. Our results showed that DDB1 is indeed required for the efficient NER of CPD, but not 6-4PP, through linkage of DDB2 DNA-binding and Cul4A E3 ligase activities. Surprisingly, we also found that DDB2 can be recruited to the damage sites in vivo in the absence of its heterodimerization with DDB1.

RNA interference. DDB1 siRNA oligonucleotides and nonspecific siRNA were synthesized by Dharmacon (Lafayette, CO) in a purified and annealed duplex form. The sequences, targeting four different regions of DDB1 gene, were as follows: duplex-1, 5′-UAACAUGAGAACUCUUGUC-3′; duplex-2, 5′-AUAAACAGCAGGUCCUUGC-3′; duplex-3, 5′-CCAUUAUCAAGGUAUGUCA-3′; duplex-4, 5′-UUAUUAUCGCGAUCUAGUG-3′. siRNA transfection was done with Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer.

Whole cell extract preparation and fractionation of cellular proteins. For whole cell extract preparation, the cells were harvested by trypsinization and lysed by boiling for 10 minutes in a sample buffer [2% SDS, 10% glycerol, 10 mmol/L DTT, 62 mmol/L Tris-HCl (pH 6.8), and protease inhibitor cocktail]. Fractionation of cellular proteins was conducted as described previously (
30). Briefly, the cells were trypsinized and resuspended in hypotonic buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 1.5 mmol/L MgCl2, and protease inhibitor cocktail] and treated with 0.1% Triton X-100 to get fraction S1, which was further centrifuged at high speed to yield the clear supernatant cytoplasmic soluble proteins. The nuclear pellet was then washed twice with isotonic sucrose buffer [50 mmol/L Tris-HCl (pH 7.4), 0.25 mol/L sucrose, 5 mmol/L MgCl2, and protease inhibitor cocktail] and treated with higher concentration of Triton X-100 (1%) in LS buffer [10 mmol/L Tris-HCl (pH 7.4), 0.2 mmol/L MgCl2, and protease inhibitor cocktail] to remove the nuclear envelope and get the fraction of nucleoplasmic soluble proteins. The nuclear pellet was further extracted consecutively with increasing concentrations (0.3, 0.5, and 2.0 mol/L) of NaCl in LS buffer to result in supernatant fractions, respectively, designated as 0.3, 0.5, and 2.0. The nuclear residue comprising of DNA and nuclear matrix was dissolved by sonication in LS buffer. Each protein fraction, corresponding to an equivalent cell number, was loaded for SDS-PAGE and analyzed by immunoblotting with indicated antibodies.

In vivo ubiquitylation. HeLa-DCH cells were first transfected with 100 nmol/L DDB1 siRNA for 24 hours and then transfected with 2 μg HA-ubiquitin–expressing plasmid for another 24 hours. MG132 (10 nmol/L) was added to the culture 0.5 hours before UV irradiation at 40 J/m2 and kept in the same medium for 2 hours. Cells were collected and the nuclei were extracted with NE buffer [20 mmol/L HEPES (pH 7.9), 25% glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, and protease inhibitor cocktail] and immunoprecipitated with anti-FLAG M2 affinity gel (Sigma, St. Louis, MO). The immunoprecipitates were subjected to Western blotting and detected with anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Western blot analysis of proteins. The proteins were quantified, separated by SDS-PAGE, and the immunoblot analysis was done by using chemiluminescent detection. The following antibodies were used: rabbit anti-DDB1 and Cul4A antibodies (kindly provided by Dr. Yue Xiong, University of North Carolina, NC; both at 1:1,000), rabbit anti-DDB2 antibody (produced by us as previously described in ref.
31; 1:1,000), rabbit anti-ubiquitin antibody (Santa Cruz Biotechnology, 1:1,000), monoclonal anti-actin (Neomarkers, Fremont, CA, 1:500).

Localized micropore UV irradiation and immunofluorescent staining. The cells growing on glass coverslips were washed with PBS and UV irradiated as described previously (
32). Briefly, an isopore polycarbonate filter (Millipore, Bedford, MA), containing pores of 5 μm in diameter, was placed on the top of the cell monolayer. The filter-covered coverslips were irradiated from above with UV-C (254 nm) at 100 J/m2. The filter was then gently removed and the cells were incubated in serum-free medium for 0.5 hours followed by fixation and permeabilization with 2% paraformaldehyde and 0.5% Triton X-100 in PBS. The cells were then double stained with mouse anti-HA (to visualize DDB2; Roche; 1:100) and rabbit anti-CPD (UV-2, generated in our lab; 1:1,000), or mouse anti-Myc (to visualize Cul4A; Roche; 1:40) and rabbit anti-CPD (UV-2) as described previously (
32). Fluorescence images were obtained with a Nikon Fluorescence Microscope 80i (Nikon, Tokyo, Japan) fitted with appropriate filters for FITC and Texas red. The digital images were then captured with a cooled charge coupled device camera and processed with the help of its SPOT software (Diagnostic Instruments, Sterling Heights, MI).

Quantitation of CPD and 6-4PP by immuno-slot-blot assay. The amounts of different photoproducts in DNA were quantified by previously established noncompetitive immuno-slot-blot assay (
29). Briefly, after UV exposure (20 J/m2) and desired incubation periods, OSU-2 cells were recovered by trypsinization and immediately lysed for DNA isolation. The same amount of DNA samples were loaded on nitrocellulose membranes and the amounts of CPD or 6-4PP were detected with monoclonal anti-CPD antibody (TDM2, 1:2,000) or monoclonal anti-6-4PP (64M-2, 1:2,000) antibody (MBL International, Co., Woburn, MA). The intensity of each band was determined by laser densitometric scanning and the amount of damage remaining, compared with the initially induced DNA damage, was used to calculate the relative repair rates.

Results

DDB1 is required for efficient NER of CPD, but not of 6-4PP, in mammalian cells. Unlike DDB2, DDB1 is vital for organism survival as there are no living individuals with a deficiency in this protein. To understand the role of DDB1 in NER, we applied RNA interference technology to knock down the expression of DDB1 in mammalian cells and analyzed the removal of UV-induced CPD and 6-4PP in these cells. First, we checked the knockdown efficiency of siRNA specific for DDB1. Four different duplexes of siRNA, targeting various sequences of DDB1 mRNA, were individually transfected into normal human fibroblast OSU-2 cells at 100 nmol/L for 48 hours. The whole cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-DDB1 antibody. As shown in
Fig. 1A
, all the DDB1 siRNA duplexes were able to knock down the expression of DDB1, albeit to varying degrees. Among them, duplex-1 siRNA exhibited the highest proficiency (>90%) to deplete the cellular DDB1 protein, and the inhibition of DDB1 expression was sustained up to 72 hours following transfection (data not shown). We also tested the ability of the RNA interference duplexes in HeLa cells and found knockdown efficiency comparable with that of OSU-2 cells (data not shown). Therefore, duplex-1 was used in all the subsequent experiments to determine the effects of DDB1 abrogation on DNA repair. OSU-2 cells were transfected with DDB1 siRNA duplex-1 for 48 hours to achieve the loss of cellular DDB1 protein, followed by UV irradiation at 20 J/m2, and a repair time up to an additional 24 hours. An aliquot (30 ng) of the DNA isolated at varying postrepair times was subject to the analysis of initial and remaining amount of lesion by immuno-slot-blot analysis with cognate anti-CPD or anti-6-4PP antibodies. As shown in
Fig. 1B and C, nonspecific siRNA-transfected OSU-2 cells were able to repair >40% CPD at 24 hours following UV irradiation, which was comparable with repair rate in untransfected cells (data not shown). However, DDB1 siRNA-transfected cells showed only a 25% repair of CPD during the same period. In contrast, the repair efficiency of 6-4PP in DDB1 siRNA-transfected cells was identical to that in nonspecific siRNA-transfected cells (
Fig. 1D and E). We also tested the effect of other three siRNA oligos on the removal of CPD and obtained similar results (data not shown). These results indicate that DDB1 protein is required for the efficient repair of CPD, but not of 6-4PP, in mammalian cells.

DDB1 is required for CPD but not 6-4PP removal. Normal human fibroblast, OSU-2 cells, were transfected with 100 nmol/L of four different DDB1-specific siRNAs for 48 hours to knock down the expression of DDB1 protein. A, cell lysates were loaded on 4% to 12% gradient SDS-PAGE. After transferring to nitrocellulose membrane, proteins were detected with anti-DDB1 and anti-actin antibodies (arrow, actin band). B to E, cells were UV irradiated at 20 J/m2 and incubated for the indicated times. Total DNA was isolated and 30 ng was loaded for slot-blot analysis. The remaining damage, CPD (B and C) and 6-4PP (D and E), in the cells was detected with mouse anti-CPD and mouse anti-6-4PP antibodies, respectively. The intensity of the bands was calculated by densitometric scanning and the repair efficiency was calculated from relative comparisons of initial and remaining DNA damage. Points (C and E), mean percentage repair of three independent measurements.

DDB1 protein translocates to damaged chromatin sites in a DDB2-dependent manner. Previous studies have shown that DDB1 is not needed for the repair of damage in the NER system reconstituted in vitro (
19,
20). However, in vivo studies have shown that ectopically expressed DDB1 could be recruited to the damage sites following UV irradiation (
33), suggesting that DDB1, as a subunit of DDB, is capable of binding to the damaged sites. In this study, we investigated the chromatin binding and UV-induced translocation of DDB1 by using cellular fractionation and immunoblot analysis. OSU-2 cells, with or without UV irradiation, were differentially lysed and the lysates were fractionated into six fractions according to their resistance to detergent or salt extraction. As shown in
Fig. 2A
, most of the DDB1 protein in OSU-2 cells resides in the 0.3 mol/L fraction, indicating that DDB1 is loosely bound to chromatin. Following UV irradiation, there is a prominent shift in DDB1 chromatin association as almost the entire amount of available DDB1 translocates to the 2.0 mol/L fraction, indicating that the protein is avidly immobilized at the UV damage sites in chromatin. It is clear that the DDB1 has nuclear localization and its weak association with undamaged chromatin becomes stronger in the presence of UV-induced damage. The fate of DDB1 protein upon irradiation in DDB2-deficient XP-E cells is shown in
Fig. 2A. Unlike DDB2-proficient OSU-2 cells, most of the DDB1 protein was localized in the cytoplasmic fraction of XP-E cells. This is consistent with the knowledge that DDB2 is required in the transfer of cytoplasmic DDB1 to the nucleus (
23). About half of the DDB1 present in the nucleus is in the loosely bound form as it was released into 0.3 mol/L fraction. Moreover, the distribution of this chromatin-bound DDB1 remained unchanged following UV irradiation of XP-E cells. This indicates that the UV-induced tight-binding of DDB1 to damaged chromatin, observed in normal cells, results from the presence of active DDB2 protein. To confirm the active role of DDB2, we investigated the UV-induced redistribution of DDB1 in another DDB2-deficient 041 cell line as well as the 041 cells with stably transfected DDB2 cDNA. 041 cells lack DDB2 because it is a p53-null cell line that fails to induce p53 downstream effector DDB2 protein (
12). As shown in
Fig. 2B, the DDB2-deficient 041 cells exhibit a DDB1 distribution pattern identical to XP-E cells and it remains unaltered following UV irradiation. On the other hand, UV irradiation results in a clear translocation of DDB1 protein to a tight-binding form in 041 cells in which DDB2 expression was ectopically restored. As part of the protein translocation experiments, we also determined the localization and fate of Cul4A within chromatin in unirradiated and UV-irradiated normal human OSU-2 cells.
Figure 2C shows that about half of the cellular Cul4A resides in cytoplasm and major portion of the nuclear Cul4A occurs in the loosely bound form of the chromatin. However, upon UV irradiation of cells, the nuclear Cul4A shows distinct translocation from the 0.3 to 2.0 mol/L fraction, indicating that UV irradiation induces tight binding of Cul4A to damaged chromatin. It is worth mentioning that Cul4A recovered in the 2.0 mol/L fraction in all these experiments represent a modified form as the corresponding bands show an easily discernable slower migration during SDS-PAGE. It is known that Cul4A can be modified by NEDD8 ubiquitin-like protein (neddylation) and this modification is proposed to stimulate the ubiquitin ligase activity (
25,
34,
35). We believe that the slow-migrating Cul4A is a neddylated protein and it is this modified form that binds to damaged chromatin tightly. Altogether, these fractionation/association results indicate that DDB1 subunit component, aided strictly by the DDB2 subunit, translocates and binds tightly to UV-damaged sites in chromatin.

UV-induced tight binding of DDB1 to damaged chromatin is DDB2-dependent. A, normal human fibroblast (OSU-2) cells and DDB2-deficient (XP-E) cells were UV irradiated at 20 J/m2 and incubated for another 30 minutes. The cells were fractionated as described in Materials and Methods. Individual protein fractions, corresponding to equivalent cell number, were subjected to SDS-PAGE and analyzed by immunoblotting with anti-DDB1 antibody. B, DDB2 and p53-deficient (041) cells, and 041 cells with stable expression of ectopic DDB2, were UV irradiated, fractionated, and analyzed by immunoblotting. The translocation of DDB1 protein was detected with anti-DDB1 antibody. C, OSU-2 cells were UV irradiated at 20 J/m2 and processed as in (A and B). The translocation of Cul4A protein was detected with anti-Cul4A antibody. S2, cytoplasmic soluble proteins; TW, nucleoplasmic soluble proteins; 0.3, proteins binding to chromatin loosely; 0.5, proteins binding to chromatin with intermediate affinity; 2.0, proteins binding to chromatin tightly; NR, nuclear residue.

DDB1 is required for UV-induced Cul4A, but not DDB2 recruitment. It is already known that DDB1, DDB2, and Cul4A form a complex with an inherent E3 ubiquitin ligase activity (
25), and three subunits of this complex can be recruited to UV-induced damage sites (
14,
33,
36). Nevertheless, the role of DDB1 in the UV-induced recruitment of Cul4A and/or DDB2 is still unclear. To address this question, we first investigated the role of DDB1 in UV-induced translocation of Cul4A and DDB2 by using RNA interference and cellular fractionation. OSU-2 cells were transfected with DDB1 siRNA or mock-transfected for 48 hours to knock down the expression of DDB1. The cells were UV irradiated at 20 J/m2 and further incubated for 30 minutes to enable the initiation of damage processing. The cellular fractionation was done as before and the nuclear fractions [0.3, 0.5, and 2.0 mol/L, and nuclear residue] were subjected to Western blotting. As shown in
Fig. 3
, in the absence of DDB1 siRNA transfection, Cul4A translocates almost quantitatively from the lower 0.3 to 2.0 mol/L fraction upon UV irradiation. Cul4A translocation is easily seen to be compromised upon DDB1 knockdown as considerable amount of cellular Cul4A still resides in 0.3 mol/L fraction following UV irradiation and only a minute amount associated with 2.0 mol/L fraction. These results indicate that DDB1 is required for tight binding of Cul4A to chromatin following UV irradiation. We also analyzed the redistribution of DDB2 following UV irradiation in OSU-2 cells with and without the knockdown of cellular DDB1. As previously reported (
30), UV irradiation induces a prompt and unambiguous translocation of DDB2, from 0.3 to 2.0 mol/L fraction, in the cells with normal DDB1 expression. But surprisingly, in the absence of DDB1 expression in cells, UV-induced DDB2 translocation from loosely bound to tightly bound form within chromatin was completely unaffected. This response, which is in obvious contrast to the movement of Cul4A component of the complex, indicates that DDB2 can bind tightly to the damaged chromatin as a monomer and that the participation of partner DDB1 protein is dispensable in this process. To confirm this unique and interesting observation, we conducted micropore local UV irradiation combined with in situ immunofluorescent staining to track the movement of Cul4A as well as DDB2 within chromatin in the native cellular environment. HeLa-DCH cells with overexpressed Myc-tagged Cul4A and FLAG-HA-tagged DDB2 were first transfected with DDB1 siRNA or mock-transfected for 48 hours. The cells growing on coverslips were UV irradiated at 100 J/m2 through a filter with pores of 5 μm diameter and then incubated for another 30 minutes to allow movement of the involved proteins. UV-irradiated cells were processed for visualization by double immunostaining with anti-Myc (Cul4A) and anti-CPD or anti-HA (DDB2) and anti-CPD antibody combinations. As shown in
Fig. 4A
, Cul4A is readily detected at the CPD DNA damage sites following UV irradiation in the cells with normal expression of DDB1. Greater than 90% damage sites exhibited the concomitant Cul4A recruitment. However, the UV-induced recruitment of Cul4A was dramatically inhibited upon DDB1 knockdown in HeLa-DCH cells as <10% damage sites showed detectable Cul4A recruitment. This result confirmed that UV-induced Cul4A recruitment to damage sites requires DDB1 protein. Meanwhile, we detected the expected recruitment of DDB2 in HeLa-DCH cells without DDB1 knockdown following UV irradiation. As seen in
Fig. 4B, DDB2 is readily detected at the sites of CPD with 90% damage sites having distinct DNA damage–specific DDB2 recruitment under conditions of normal DDB1 expression. Consistent with chromatin association results above, eliminating DDB1 from cells had no perceivable effect on in situ recruitment of DDB2 to damage sites. Greater than 90% damage-specific DDB2 sites were seen in the absence or presence of DDB1 knockdown. The data reinforced the contention that UV-induced recruitment of DDB2 to the damage sites does not require DDB1. In essence, these results help conclude that DDB2 can be recruited to UV-induced damage sites as an independent component, whereas Cul4A recruitment occurs only in the presence of DDB1, most likely as part of the complex.

Knockdown of DDB1 protein compromises UV-induced Cul4A but not DDB2 translocation within chromatin. OSU-2 cells were transfected with 100 nmol/L siRNA specific for DDB1 or mock transfected for 48 hours. The cells were UV irradiated at 20 J/m2, further incubated for 30 minutes, and then subjected to cellular fractionation as described in Materials and Methods. The fractions obtained from nuclei (0.3, 0.5, 2.0, and nuclear residue) were loaded for SDS-PAGE and analyzed by Western blotting with anti-Cul4A and anti-DDB2 antibodies.

Knockdown of DDB1 protein blocks UV-induced in situ recruitment of Cul4A but not DDB2 to damage sites. HeLa-DCH cells, grown on coverslips, were UV irradiated at 100 J/m2 through a 5 μm isopore filter and maintained in fresh medium for 30 minutes. The cells were fixed and double immunostained with anti-Myc (Cul4A) and anti-CPD antibodies (A) or anti-HA (DDB2) and anti-CPD antibodies (B), as described in Materials and Methods. The total number of Cul4A or DDB2 foci and CPD foci were counted from at least five separate fields. The ratios of Cul4A to CPD or DDB2 to CPD were calculated from the numbers for independent fluorescent foci.

DDB1 participates in NER through the ubiquitylation and degradation of DDB2. It has been shown that DDB2 can be ubiquitylated by DDB-Cul4A E3 ubiquitin ligase in vivo and in vitro (
27,
28,
37,
38). Besides, it is also known that Cul4A itself has E3 activity and that DDB1 acts as an adaptor for Cul4A to ubiquitylate its substrates, e.g., CDT1 (
26). However, the role of DDB1 in the ubiquitylation and degradation of DDB2 has not been established. To address this question, we first investigated the UV-induced DDB2 degradation in the absence of DDB1 protein. As shown in
Fig. 5A
, knockdown of DDB1 expression by its specific siRNA abolished the key UV-induced early response, i.e., prominent degradation of DDB2 protein. This indicated that the presence of DDB1 is a key requirement for UV-induced DDB2 degradation in irradiated cells. Next, we analyzed the effect of DDB1 on UV-induced DDB2 ubiquitylation in vivo. HeLa-DCH cells were transfected with DDB1 siRNA or mock-transfected for 24 hours and then transfected with HA-tagged ubiquitin expression construct for another 24 hours. Proteasome inhibitor MG132 was added to the cultures 1 hour before UV irradiation and kept in the cultures during the period of repair to ensure the detection of ubiquitylated proteins. The cell lysates was immunoprecipitated with anti-FLAG M2 affinity gel and the immunoprecipitates were detected by Western blotting with anti-ubiquitin antibody. As shown in
Fig. 5B, UV irradiation enhanced the overall cellular ubiquitylation of DDB2 in the cells normally expressing DDB1 protein (lane 3 versus lane 2). However, upon DDB1 knockdown, not only the physiologic, but also the UV-induced, ubiquitylation of DDB2 was significantly inhibited (lane 4 versus lane 2 and lane 5 versus lane 3). These data indicated that DDB1 is an important adaptor component for Cul4A to execute its E3 ligase activity to ubiquitylate the target substrate, DDB2.

Knockdown of DDB1 comprises UV-induced DDB2 ubiquitylation and degradation. A, HeLa-DCH cells were transfected with DDB1 siRNA or mock transfected for 48 hours. The cells were UV irradiated at 20 J/m2 and incubated for another 2 hours. The cell lysates were subjected to SDS-PAGE and detected with anti-DDB1, anti-DDB2, and anti-actin antibodies. B, HeLa-DCH cells were first transfected with 100 nmol/L DDB1 siRNA or mock-transfected for 24 hours and then transfected with 2 μg HA-tagged ubiquitin for another 24 hours. MG132 (10 nmol/L) was added to the cultures for 30 minutes; the cells were UV irradiated at 40 J/m2 and kept in the same medium for 2 hours. The cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel, the immunoprecipitates were subjected to immunoblotting, and the Western blots were detected with anti-ubiquitin antibody.

Discussion

Presence of DDB2 protein is critical for DDB1 to help initiate NER. In this study, we have provided evidence for the direct participation of DDB1 in NER of CPD, which is known to repair at a slower rate in different kinds of mammalian cells. However, DDB1 was not needed for the removal of another major photolesion, 6-4PP, which is rapidly eliminated from DNA of living organisms. This differential lesion-specific role of DDB1 in NER is functionally similar to that of DDB2 (
12,
30,
39). This is not surprising as the underlying mechanism to initiate productive repair is based on the binding of the heterodimeric protein to DNA damage. Earlier studies have reported that DDB2 is required for the recruitment of exogenously transfected DDB1 to the local UV-induced damage sites (
33). Here, we tested the chromatin binding of endogenous DDB1 protein following UV irradiation and show that almost all the native DDB1 in the nuclei translocates from loosely bound to a tightly bound chromatin form upon UV irradiation. The DDB1 translocation mimics the UV-induced response observed with native cellular DDB2 (
30). Nevertheless, DDB1 translocation to damage is strictly dependent on the presence of functional DDB2 protein. For example, UV-induced translocation of DDB1 failed to occur in two different DDB2-deficient XP-E and 041 cells. However, restoration of DDB2 in 041 cells, by the ectopic expression, also restored the normal translocation of DDB1. In essence, the data suggest that DDB1 is able to functionally participate in NER only upon its intimate physical interaction with DDB2 protein component.

DDB and Cul4A cooperate in initiating NER. Several studies have shown that restoration of DDB2 in DDB2-deficient cells enhanced the efficiency of CPD removal (
14,
30,
39). In addition, our recent work has shown that Cul4A is required for global genomic repair of CPD but not of 6-4PP (
40). In the present work, we show that DDB1, as a subunit of DDB-Cul4A E3 ligase, independently contributes to the efficient global genomic repair of CPD but not of 6-4PP. It is established that a defect in any one of three major subunits of DDB-Cul4A complex could impair its E3 ligase activity (
26,
36). Therefore, the requirement of DDB-Cul4A E3 ligase activity for initiating efficient NER could be affected by selective abrogation of any of these components as DDB1, DDB2, and Cul4A must work cooperatively to participate in NER. To date, the known NER-related substrates of DDB-Cul4A E3 ligase include DDB2 itself, XPC, and histone H2A (
27,
28,
36–
38). DDB2 can be ubiquitylated by DDB-Cul4A E3 ligase in vivo as well as in vitro (
27,
28,
37,
38). Upon ubiquitylation, DDB2 is promptly degraded inside the cell via proteasome (
39–
41). Moreover, evidence is now fast accumulating in support of the critical role of ubiquitylation and degradation of DDB2 for the necessary recruitment of XPC to the damage sites. First, Sugasawa et al. (
37) have shown that UV-DDB seems to lose its high binding affinity for 6-4PP when DDB2 is extensively polyubiquitylated in vitro. Second, the inhibition of DDB2 degradation, by treatment with proteasome inhibitor MG132 or by knockdown of Cul4A, severely affects the recruitment of XPC to DNA damage sites in vivo (
40,
42). Therefore, after recognition of damaged DNA (e.g., CPD), DDB is thought to act in the physical handover of DNA lesions to the next repair protein, XPC, for assembling the global genomic repair machinery. In the process, DDB2 component is promptly eliminated via proteasomal degradation. Interestingly, following irradiation, XPC too is ubiquitylated upon arrival at the damage sites (
31,
37). However, the similar ubiquitylated state of XPC, although mediated by the same DDB-Cul4A E3 ligase in vitro, does not trigger its degradation. The significance of this differential fate of two closely related factors is not entirely clear. It should be noted that polyubiquitylated XPC in a cell-free system displays a higher DNA-binding activity compared with its unmodified form (
37). Thus, these in vitro binding experiments seem to suggest that XPC ubiquitylation could be playing a role in the regulation of factor assembly at DNA damage sites to achieve the efficient NER. A recent study seems to show that DDB-Cul4A E3 ligase actually targets histone H2A protein for monoubiquitylation at the UV damage sites (
36). Given that the ubiquitylation of H2A is related to the condensation of chromatin (
43), this finding suggests that DDB-Cul4A E3 ligase might contribute to NER by opening the condensed chromatin for making the buried damage accessible to additional factors needed for completing NER. The emerging model indicates a close coordination of the activities of DDB1, DDB2, and Cul4A components to function in NER within the chromatin context. Among the different complex components, DDB2 has intrinsic DNA-binding activity (
44), Cul4A possesses E3 ligase activity, whereas DDB1 interacts with both of them to function as an essential bridge to link damaged DNA-binding to ubiquitylation activity. In essence, through the DDB1 adaptor molecule, DDB2 physically recruits the E3 ligase, to the damaged chromatin sites, for the ubiquitylation of NER-related proteins, including itself.

DDB2 can bind to damaged DNA as a monomer. Although DDB has been studied extensively, there is no clear agreement whether both the DDB1 and DDB2 subunit components are required for DNA binding. Early on, it was suggested that DDB1, which might get activated in a catalytic manner (“hit-and-run”) by DDB2 (
45), possesses damage-specific, DNA-binding activity (
46). Another study has indicated that only the DDB1-DDB2 heterodimeric complex could bind to damaged DNA (
8). Recently, Kulaksiz et al. (
44) tested recombinant DDB1 and DDB2 for binding activity in vitro and found that the damaged DNA-binding property of DDB is conferred by the DDB2 subunit. However, the results from Wittschieben et al. (
47) showed that binding activity of DDB is reconstituted only when the two subunits are mixed together; both subunits are therefore necessary and sufficient for binding to the DNA substrate. To address this question in vivo, we tested the recruitment of DDB2 to the damaged chromatin in the absence of DDB1 by using two different assays, e.g., movement upon chromatin association and recruitment in locally micropore UV-irradiated sites. Both assays were consistent in revealing that DDB2 can translocate to damage DNA unaided by DDB1. Thus, the presence of DDB1 does not seem to be a requirement for simple damage-specific binding activity of DDB2 in chromatin. Nevertheless, without DDB1 in the DDB complex, and as discussed above, E3 ligase cannot locate to damage sites to accomplish the initial processing steps of global genomic repair.

DDB1 could act in NER through alternate mechanisms. In mammalian cells, DDB1 or DDB1-like proteins have been found in a significant number of complexes in association with proteins other than DDB2. Martinez et al. (
48) have reported that DDB1 interacts with STAGA (SPT3-TAFII31-GCN5L acetylase) complex, which contains a histone acetyltransferase, GCN5. The interaction of UV-DDB with STAGA might target the nucleosome acetylase activity of GCN5L to damaged chromatin sites to facilitate the assembly and/or function of the NER machinery on nucleosomes, again supporting a role of UV-DDB in DNA repair within chromatin. Another protein, SAP130, with sequence similarity to DDB1 has been found in the TBP-free TAFII complex (TFTC). TFTC preferentially binds UV-irradiated DNA and preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA, suggesting that UV-damaged, DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation (
49). In addition, Datta et al. (
50) have shown that DDB associates with the CBP/p300 family of proteins, in vivo and in vitro, suggesting that DDB participates in global genomic repair by recruiting CBP/p300 to the damaged-chromatin. Rapic-Otrin et al. (
41) has further provided evidence about the interaction between p300 and DDB occurring through the DDB1 subunit, and independent of DDB2. It is quite possible that the histone acetyltransferase activities of the CBP/p300 proteins induce chromatin remodeling at the damaged sites to allow recruitment of the repair complexes. Thus, DDB1 is a versatile cellular factor capable of participating in NER, mainly by bringing together essential components from different complexes to the sites of damaged chromatin for the purpose of opening the condensed chromatin structure to allow access to NER machinery.

Acknowledgments

Grant support: NIH grants ES2388, ES12991, and CA93413 (A.A. Wani).

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